Peter Mancuso

The prevalence of obesity and metabolic diseases have risen significantly over the past decades. Chronic inflammation in obesity is a link between obesity and secondary disease. While macrophages and monocytes are known to contribute to metabolic disease risk during diet exposure, little is known about the contribution of neutrophils. We assessed the impact of obesity on neutrophils using a 16-week model of diet-induced obesity. Bone marrow (BM) neutrophils significantly expanded with chronic high-fat diet (HFD), significantly decreased TNFɑ protein release, and impaired neutrophil regenerative function compared to normal diet (ND) neutrophils. scRNAseq and flow cytometry demonstrated HFD neutrophil heterogeneity and validated that these cells do not have elevated expression of proinflammatory genes without secondary stimulation. HFD neutrophils showed elevated expression of genes associated with lipid metabolism—acyl-CoA thioesterase 1 (Acot1), carnitine palmitoyltransferase 1a (Cpt1a), and perilipin 2 (Plin2). Consistent with the importance of lipid metabolism in driving dysfunction, neutrophils from HFD-fed animals and neutrophils treated with palmitate had impaired bacterial phagocytosis and killing responses. These data shed light on the complex regulation of intracellular lipids and the role of metabolism on neutrophil function during homeostasis and disease.

Obesity impairs host defense against Klebsiella pneumoniae, but responsible mechanisms are incompletely understood. To determine the impact of diet-induced obesity on pulmonary host defense against K. pneumoniae, we fed 6-wk-old male C57BL/6j mice a normal diet (ND) or high-fat diet (HFD) (13% vs. 60% fat, respectively) for 16 wk. Mice were intratracheally infected with Klebsiella, assayed at 24 or 48 h for bacterial colony-forming units, lung cytokines, and leukocytes from alveolar spaces, lung parenchyma, and gonadal adipose tissue were assessed using flow cytometry. Neutrophils from uninfected mice were cultured with and without 2-deoxy-d-glucose (2-DG) and assessed for phagocytosis, killing, reactive oxygen intermediates (ROI), transport of 2-DG, and glucose transporter (GLUT1–4) transcripts, and protein expression of GLUT1 and GLUT3. HFD mice had higher lung and splenic bacterial burdens. In HFD mice, baseline lung homogenate concentrations of IL-1β, IL-6, IL-17, IFN-γ, CXCL2, and TNF-α were reduced relative to ND mice, but following infection were greater for IL-6, CCL2, CXCL2, and IL-1β (24 h only). Despite equivalent lung homogenate leukocytes, HFD mice had fewer intraalveolar neutrophils. HFD neutrophils exhibited decreased Klebsiella phagocytosis and killing and reduced ROI to heat-killed Klebsiella in vitro. 2-DG transport was lower in HFD neutrophils, with reduced GLUT1 and GLUT3 transcripts and protein (GLUT3 only). Blocking glycolysis with 2-DG impaired bacterial killing and ROI production in neutrophils from mice fed ND but not HFD. Diet-induced obesity impairs pulmonary Klebsiella clearance and augments blood dissemination by reducing neutrophil killing and ROI due to impaired glucose transport.

Background: The menopausal transition is characterized by increased cardiovascular risk, weight gain, and increased adiposity for many women. The adipose‐derived secretory proteins adiponectin and leptin are associated with insulin resistance, metabolic syndrome, and cardiovascular disease but their role in subclinical atherosclerotic disease is unclear. This cross‐sectional study evaluated the associations of adiponectin and leptin with carotid artery intima‐media thickness, adventitial diameter, presence of carotid plaques, and brachial‐ankle pulse wave velocity (baPWV) in women aged 54 to 65 years. Methods and Results: Participants were 1399 women from SWAN (Study of Women's Health Across the Nation), a community‐based study of women transitioning through menopause. Carotid ultrasound and baPWV measures were obtained at SWAN follow‐up visits 12 or 13, when 97% of participants were post‐menopausal. Adipokines were assayed from serum specimens obtained concurrently at these visits. Linear and logistic regression models were used to evaluate adiponectin or leptin, both log‐transformed attributable to skewness, in relationship to carotid artery intima‐media thickness, adventitial diameter, baPWV, and presence of carotid plaque. Covariates included age, race, study site, smoking, alcohol use, obesity, cardiovascular disease risk factors, and menopausal status. Lower levels of adiponectin were related to greater carotid artery intima‐media thickness, wider adventitial diameter, and faster baPWV; associations were attenuated after adjusting for cardiovascular disease risk factors. Higher levels of leptin were associated with greater carotid artery intima‐media thickness and wider adventitial diameter in minimally and fully adjusted models, and contrary to expectation, with slower baPWV, particularly among women with diabetes mellitus or obesity. Conclusions: Adiponectin and leptin are 2 important inflammatory pathways that may contribute to adverse subclinical cardiovascular disease risk profiles in women at midlife.

Extracellular vesicles (EVs) contain a diverse array of molecular cargoes that alter cellular phenotype and function following internalization by recipient cells. In the lung, alveolar macrophages (AMs) secrete EVs containing suppressor of cytokine signaling 3 (SOCS3), a cytosolic protein that promotes homeostasis via vesicular transfer to neighboring alveolar epithelial cells. Although changes in the secretion of EV molecules—including but not limited to SOCS3—have been described in response to microenvironmental stimuli, the cellular and molecular machinery that control alterations in vesicular cargo packaging remain poorly understood. Furthermore, the use of quantitative methods to assess the sorting of cytosolic cargo molecules into EVs is lacking. Here, we utilized cigarette smoke extract (CSE) exposure of AMs as an in vitro model of oxidative stress to address these gaps in knowledge. We demonstrate that the accumulation of reactive oxygen species (ROS) in AMs was sufficient to augment vesicular SOCS3 release in this model. Using nanoparticle tracking analysis (NTA) in tandem with a new carboxyfluorescein succinimidyl ester (CFSE)-based intracellular protein packaging assay, we show that the stimulatory effects of CSE were at least in part attributable to elevated amounts of SOCS3 packaged per EV secreted by AMs. Furthermore, the use of a 20S proteasome activity assay alongside treatment of AMs with conventional proteasome inhibitors strongly suggest that ROS stimulated SOCS3 release via inactivation of the proteasome. These data demonstrate that tuning of AM proteasome function by microenvironmental oxidants is a critical determinant of the packaging and secretion of cytosolic SOCS3 protein within EVs.

During the last 40 years, there has been a world-wide increase in both the prevalence of obesity and an increase in the number of persons over the age of 60 due to a decline in deaths from infectious disease and the nutrition transition in low and middle income nations. While the increase in the elderly population indicates improvements in global public health, this population may experience a diminished quality of life due to the negative impacts of obesity on age-associated inflammation. Aging alters adipose tissue composition and function resulting in insulin resistance and ectopic lipid storage. A reduction in brown adipose tissue activity, declining sex hormones levels, and abdominal adipose tissue expansion occur with advancing years through the redistribution of lipids from the subcutaneous to the visceral fat compartment. These changes in adipose tissue function and distribution influence the secretion of adipose tissue derived hormones, or adipokines, that promote a chronic state of low-grade systemic inflammation. Ultimately, obesity accelerates aging by enhancing inflammation and increasing the risk of age-associated diseases. The focus of this review is the impact of aging on adipose tissue distribution and function and how these effects influence the elaboration of pro and anti-inflammatory adipokines.

Leptin is a pleiotropic hormone produced by white adipose tissue that regulates appetite and many physiological functions, including the immune response to infection. Genetic leptin deficiency in humans and mice impairs host defenses against respiratory tract infections. Since leptin deficiency is associated with obesity and other metabolic abnormalities, we generated mice that lack the leptin receptor (LepRb) in cells of the myeloid linage (LysM-LepRb-KO) to evaluate its impact in lean metabolically normal mice in a murine model of pneumococcal pneumonia. We observed higher lung and spleen bacterial burdens in LysM-LepRb-KO mice following an intratracheal challenge with Streptococcus pneumoniae. Although numbers of leukocytes recovered from bronchoalveolar lavage fluid did not differ between groups, we did observe higher levels of pulmonary IL-13 and TNFα in LysM-LepRb-KO mice 48 h post infection. Phagocytosis and killing of ingested S. pneumoniae were also impaired in alveolar macrophages (AMs) from LysM-LepRb-KO mice in vitro and were associated with reduced LTB₄and enhanced PGE₂synthesis in vitro. Pretreatment of AMs with LTB₄and the cyclooxygenase inhibitor, indomethacin, restored phagocytosis but not bacterial killing in vitro. These results confirm our previous observations in leptin-deficient ( ob/ob) and fasted mice and demonstrate that decreased leptin action, as opposed to metabolic irregularities associated with obesity or starvation, is responsible for the defective host defense against pneumococcal pneumonia. They also provide novel targets for therapeutic intervention in humans with bacterial pneumonia.

Preservation of gas exchange mandates that the pulmonary alveolar surface restrain unnecessarily harmful inflammatory responses to the many challenges to which it is exposed. These responses reflect the cross-talk between alveolar epithelial cells (AECs) and resident alveolar macrophages (AMs). We recently determined that AMs can secrete suppressor of cytokine signaling (SOCS) proteins within microparticles. Uptake of these SOCS-containing vesicles by epithelial cells inhibits cytokine-induced STAT activation. However, the ability of epithelial cells to direct AM release of SOCS-containing vesicles in response to inflammatory insults has not been studied. In this study, we report that SOCS3 protein was elevated in bronchoalveolar lavage fluid of both virus- and bacteria-infected mice, as well as in an in vivo LPS model of acute inflammation. In vitro studies revealed that AEC-conditioned medium (AEC-CM) enhanced AM SOCS3 secretion above basal levels. Increased amounts of PGE₂ were present in AEC-CM after LPS challenge, and both pharmacologic inhibition of PGE₂ synthesis in AECs and neutralization of PGE₂ in AEC-CM implicated this prostanoid as the major AEC-derived factor mediating enhanced AM SOCS3 secretion. Moreover, pharmacologic blockade of PGE₂ synthesis or genetic deletion of a PGE₂ synthase similarly attenuated the increase in bronchoalveolar lavage fluid SOCS3 noted in lungs of mice challenged with LPS in vivo. These results demonstrate a novel tunable form of cross-talk in which AECs use PGE₂ as a signal to request SOCS3 from AMs to dampen their endogenous inflammatory responses during infection.